CN110938656A - Recombinant expression vector of giant panda follicle-stimulating hormone, expression system and preparation method - Google Patents

Abstract

The invention discloses a carrier, an expression system and a preparation method for recombinant expression of giant panda follicle stimulating hormone, which relate to the technical field of genetic engineering and comprise a first gene for coding a giant panda FSH α subunit and a second gene for coding a giant panda FSH β subunit, wherein the sequence of the first gene is shown as SEQ ID NO.1, and the sequence of the second gene is shown as SEQ ID NO. 2.

Description

Recombinant expression vector of giant panda follicle-stimulating hormone, expression system and preparation method

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a vector for recombinant expression of giant panda follicle-stimulating hormone, an expression system and a preparation method.

Background

The breeding rate of pandas is generally improved by adopting a method of xenogenic gonadotropins, and the long-term use of xenogenic gonadotropins to induce the oestrus and ovulation of animals has been shown to cause adverse reactions to the animals, the oestrus effect is reduced, and the breeding rate of the pandas is seriously influenced. At present, no effective method can solve the technical problems.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a vector for recombinant expression of giant panda follicle-stimulating hormone, an expression system and a preparation method thereof so as to solve the technical problems.

The invention is realized by the following steps:

a carrier for recombinant expression of giant panda follicle stimulating hormone comprises a first gene for encoding a giant panda FSH α subunit and a second gene for encoding a giant panda FSH β subunit, wherein the sequence of the first gene is shown as SEQ ID NO.1, and the sequence of the second gene is shown as SEQ ID NO. 2.

Follicle Stimulating Hormone (FSH), which has a molecular weight of approximately 33KD, consists of a heterodimer formed by α and β subunits.

Among them, α subunit is composed of 120 amino acid residues, α subunit has an amino acid sequence shown in SEQ ID NO.4, β subunit is composed of about 129 amino acid residues, β subunit has an amino acid sequence shown in SEQ ID NO.5, α subunit has high conservation among species, β subunit has species specificity, α subunit and β subunit can exert FSH effect only when they are combined together.

The effects of FSH in the ovary include: promote the development of ovarian epithelial tissues, gonads and the like, promote the growth and development of follicles, promote the synthesis and secretion of estrogen (E) and estradiol (E2), and participate in the selection and further development of dominant follicles in ovaries.

The effects of FSH in the testis include: stimulates the development of testicular seminiferous tubule epithelial cells, promotes the expression regulation of male hormone, and secretes inhibin, which plays an important role in the normal formation, production and maturation of sperms. In addition, FSH may also affect the spermatogenic function of the testes.

In order to solve the problem of heterologous gonadotropins, the inventors found that two subunits (α subunit and β subunit) are expressed by using independent vectors, and the expression amount of the two subunits is mismatched during subsequent transfection, so that active folded protein cannot be obtained.

Because FSH α subunit and FSH β subunit of pandas are glycosylated to some extent, the FSH protein with the target activity cannot be expressed by adopting a prokaryotic expression method.

The invention provides a carrier for recombinant expression of giant panda follicle stimulating hormone, which comprises a first gene for coding a giant panda FSH α subunit and a second gene for coding a giant panda FSH β subunit, wherein the carrier can be used for stable and continuous active expression of the giant panda FSH α subunit and the giant panda FSH β subunit.

In a preferred embodiment of the present invention, the carrier further comprises a connecting element.

In an alternative embodiment, the linking element is a T2A element, and the sequence of the T2A element is shown in SEQ ID No. 3. The amino acid sequence of the T2A element is shown by reference to SEQ ID NO. 6.

The T2A sequence has the advantages of short sequence, easy operation, good upstream and downstream gene expression balance and the like in the process of constructing the polycistronic vector, and the FSH α and FSH β co-expression vector constructed by utilizing the T2A sequence can be translated normally after being transferred into CHO-K1 cells, in addition, T2A can play a self-cleavage function in CHO-K1 cells.

A construction method of a vector for recombinant expression of giant panda follicle stimulating hormone comprises the following steps: a first gene and a second gene are synthesized.

The first gene and the second gene respectively code FSH α subunit and FSH β subunit of panda, and two ends of the T2A element are respectively connected with the first gene and the second gene.

In an alternative embodiment, the method of construction further comprises ligating the synthesized first and second genes and the T2A element into a plasmid pCDH to construct a recombinant vector expressing panda follitropin containing the inserted sequences of the first gene, the T2A element and the second gene.

The construction method is that the plasmid pCDH is firstly carried out EcoR I and BamH I double digestion to linearize the vector, then EcoRI and BamH I double digestion is carried out to linearize the insert, and the linearized insert is connected to the linearized pCDH by T4 DNA ligase enzyme.

The first gene, the T2A element and the second gene are synthesized together and directly connected to a pcDH vector through whole enzyme digestion, and the two ends of the synthesized first gene, the T2A element and the second gene are provided with EcoR I and BamH I restriction enzyme sites.

A eukaryotic cell expression system containing a recombinant panda follicle stimulating hormone expression vector or a vector constructed by the construction method. The eukaryotic cell expression system is constructed by transferring a vector into a eukaryotic cell.

The recombinant vector for expressing the giant panda follicle stimulating hormone can be transferred into eukaryotic cells to express and produce glycosylated FSH α subunit, FSH β subunit and subunit compound thereof.

In an alternative embodiment, the eukaryotic cell is constructed from CHO-K1 cells. In other embodiments, the class of eukaryotic cells can be selected as desired.

A method for producing recombinant panda follicle stimulating hormone in a eukaryotic cell expression system comprises culturing the eukaryotic cell expression system.

In an alternative embodiment of the invention, the constructed vector for recombinant expression of giant panda follitropin is transferred into CHO-K1 cells to construct a eukaryotic cell expression system containing the vector for recombinant expression of giant panda follitropin.

In order to improve the infection efficiency and success rate, the CHO-K1 cells are infected by a lentivirus expression system. The lentiviral expression system was selected from a three plasmid system comprising the lentiviral backbone vector pCDH and the packaging plasmids psPAX2 and pmd2. g.

The vector-CMV-MCS-EF 1-Puro for recombinant expression of giant panda follicle stimulating hormone and the slow virus packaging plasmid are co-transferred into HEK293T cells, the recombinant slow virus is collected and purified and the CHO-K1 cells are infected, and a CHO-K1 cell stable expression system containing the vector for expression of the recombinant giant panda follicle stimulating hormone is constructed.

The vector for recombinant expression of giant panda follicle-stimulating hormone can be integrated on the eukaryotic cell genome through lentivirus, and long-time stable expression of a target gene can be realized.

The expression system provided by the invention can secrete the panda-derived FSH into a DMEM low-sugar medium and has activity.

The culture method of CHO-K1 cells containing the vector for recombinant expression of giant panda follicle stimulating hormone comprises the following steps:

(1) the cultured CHO-K1 cells are passaged once every 3 days, when the cell density reaches about 80%, a DMEM low-sugar culture medium is sucked out by a disposable suction tube, PBS is slowly added to submerge the cells, and a 90-mm culture dish is washed by slight shaking; (2) removing the culture medium by suction, adding appropriate amount of pancreatin/EDTA digestive juice (0.25%), incubating at 37 deg.C for 2 min; (3) adding a proper amount of culture medium into a culture dish, blowing and washing the culture dish along different directions by using a disposable suction tube to ensure that cells are eliminated from the plate as much as possible, and transferring the mixed solution into a 15ml sterile centrifuge tube; (4) gently blowing and beating the cells by using a pipette to resuspend the cells, and centrifuging the cells for 3min at 1500rpm of a desk centrifuge; (5) removing the supernatant, adding 5ml of culture medium, gently blowing and beating the resuspended cells, and centrifuging at 1500rpm for 3 min; (6) removing the supernatant, adding 5ml of culture medium, gently blowing and resuspending, centrifuging at 1500rpm for 3min, and simultaneously preparing a proper amount of complete culture medium containing 10% Fetal Bovine Serum (FBS); (7) the supernatant was removed, 10ml of complete medium was added, the cells were gently tapped and resuspended, transferred to a 90-mm petri dish and incubated in a carbon dioxide incubator (37 ℃, 5% CO 2).

In an alternative embodiment of the present invention, the above method further comprises isolating and purifying the cultured eukaryotic cell expression system.

In an alternative embodiment of the present invention, the above method for separation and purification comprises subjecting the cultured eukaryotic cell expression system to at least one of salting out, ion exchange chromatography separation and affinity chromatography separation.

The invention has the following beneficial effects:

the invention provides a vector, an expression system and a preparation method for recombinant expression of giant panda follicle stimulating hormone, which can realize the stable and continuous active expression of the FSH α subunit, the FSH β subunit and the subunit compound of the subunit of the FSH α subunit and the FSH β subunit through constructing the vector and the expression system for recombinant expression of the giant panda follicle stimulating hormone.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a gel diagram of the PCR product of mouse FSH in example 3;

FIG. 2 is a graph showing the results of fluorescence of CHO-K1 cells treated with different concentrations of recombinant human FSH protein (standard) for 6h after transfection in example 3;

FIG. 3 is a graph showing the fluorescence results of FSHR cells of mice treated with different concentrations of recombinant panda FSH protein in example 4 for 6 h.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The present example provides a method for constructing an expression vector of pCDH-FSH β -T2A-FSH α.

The construction materials used were as follows:

the strain is as follows: CHO-K1 cells and HEK293T cells, purchased from ATCC, USA; vector pcDNA3.1(+), available from Invitrogen, USA; the vector pCDH-CMV-MCS-EF1-Puro was purchased from SBI, USA; lentiviral systems packaging plasmid psPAX2+ pMD2.G was purchased from Invitrogen, USA.

Reagent: the first gene, the second gene and the T2A sequence are synthesized by Suzhou Jinweizhi biological technology, Inc., and the primer is synthesized by Beijing Liu-Hua big gene technology, Inc.; the restriction enzyme and ligase are selected from Baoriji technology (Beijing) Ltd.

The construction method comprises the following steps:

1. enzyme digestion and ligation reaction: double digestion reaction of the synthesized insert and expression vector pCDH plasmid (SBI, USA) with restriction enzymes EcoR I and BamH I; purifying and recovering the enzyme digestion product obtained by agarose gel electrophoresis separation according to the specification of the Tiangen DNA gel recovery kit; performing ligation reaction on the enzyme digestion product according to a system of a T4 ligase specification; the ligation products were transformed into competent cells and cultured in an inverted state at 37 ℃ for 12-16 h.

2. Screening positive clones: colony PCR amplification is carried out by using a universal primer expressing a vector pCDH and the colony obtained in the previous step as a template according to a system of a Taq polymerase instruction. Electrophoresis is used for detecting whether the amplification product is correct. And inoculating the positive colonies into an LB liquid culture medium, and culturing in a constant temperature shaking table to an appropriate OD value.

3. Extracting recombinant plasmids and carrying out enzyme digestion: plasmid extraction is carried out by adopting a small Tiangen plasmid extraction kit according to the instruction, and EcoR I and BamH I restriction enzymes are selected for the obtained plasmid to identify whether the size of the recombinant plasmid insert fragment is correct.

4. Plasmid sequencing and sequencing results analysis: the recombinant plasmid with correct enzyme digestion identification is selected and sent to Beijing Hua Dabiologies company for sequencing, and the sequence of the recombinant plasmid is compared and analyzed by software.

Example 2

This example provides a method for constructing CHO-K1 cell expression system.

1. HEK293T cells (ATCC company, usa) were cultured in 6-well plates and when the cells grew to 80%, transfection was performed.

2. 200 μ l of the plasmid pCDH-FSH β -T2A-FSH α (1.4 μ g) and the packaging plasmids psPAX2(1.4 μ g) and pMD2.G (0.7 μ g) were added

In buffer, vortex for 10s and then centrifuge instantaneously.

3. Adding 3 μ l

Transfection reagents (Dake, Inc., USA), vortex for 10s, instantaneous centrifugation, and room temperature (22-26℃) standing for 10 min.

4. 200 μ l of transfection mixture was added dropwise to a 6-well plate and shaken to evenly distribute the reagents.

After 5.4 hours the medium was changed and cells were grown in a growth medium containing antibiotics and 10% serum and cultured in a 5% CO2 incubator at 37 ℃.

6. At 48 h post-transfection, the culture broth was collected into 15-ml sterile, covered conical centrifuge tubes, filtered through a 0.45 μ M filter membrane, centrifuged at 3000g for 15 min at room temperature, and the supernatant containing the recombinant virus was transferred into fresh centrifuge tubes.

7. The culture medium in the CHO-K1 cell culture dish with a normal culture density of approximately 70% was aspirated, and the virus-containing supernatant described above was added to CHO-K1 cells (ATCC company, USA).

8. After 48 hours of infection, the complete culture medium containing 10 mug/ml puromycin is replaced for continuous culture, and fresh puromycin culture solution is replaced every few days according to the cell state, so that the recombinant cell line can be used for active protein preparation and subsequent functional verification experiments.

Example 3

This example provides a pGL3-CRE-luciferase reporter system for the detection of recombinantly expressed FSH active protein in example 2.

To date, annotation of the Panda genome (Panda release 92: ailMel1) has revealed that many genes, including the Panda follicle stimulating hormone receptor gene (FSHR), etc., are to be further annotated. In view of the dependence of panda Follicle Stimulating Hormone (FSH) on binding to its specific receptor

(FSHR) exerts biological effects, therefore, in the embodiment, the mouse FSHR gene is cloned, and based on the characteristic that both giant panda FSHR and mouse FSHR encode receptor protein with 692 amino acids and have 85% amino acid sequence homology, the invention uses the mouse FSHR in expression and construction of eukaryotic expression vector (pcDNA3.1-FSHR), so as to realize detection of the activity of recombinant FSH protein.

The detailed operation is as follows: ovarian tissue of a C57BL/6 mouse (Duoduosho laboratory animals Co., Ltd.) was obtained by dissection, ground with liquid nitrogen, total RNA was extracted using RNAzol reagent and reverse transcription was performed to prepare a cDNA template, and finally the mouse FSHR gene was amplified by PCR using a mouse ovarian tissue cDNA library as a template to obtain a PCR product with a total length of about 2079bp and an electrophoresis gel diagram of the mouse FSH gene as shown in FIG. 1. After being digested by BamH I and EcoR I, the DNA is connected to a eukaryotic expression vector pcDNA3.1 (Invitrogen corporation, USA), and whether a mouse FSHR gene is connected to the expression vector (pcDNA3.1+ FSHR) is verified by sequencing.

The cDNA sequence of the mouse FSHR gene cloned by the research is shown by referring to SEQ ID NO.7, and the amino acid sequence is shown by referring to SEQ ID NO. 8.

It has been shown that FSH hormone-dependent activation of its specific FSHR receptor relies on the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway to alter the expression profile of downstream target proteins and exert physiological functions.

In this example, a mouse FSHR expression plasmid (pcDNA3.1+ FSHR) and a pGL3-CRE-luciferase reporter plasmid (Promega, USA) were co-transfected into CHO-K1 cells, the transfected CHO-K1 cells were treated with 0.1ng/ml, 1ng/ml and 10ng/ml recombinant human FSH proteins (Standard, RD, USA) for 6h, lysed and incubated with added substrate, and the effect of LH hormone on the intracellular cAMP signaling pathway was determined by measuring the fluorescence readings using a microplate reader.

The cell transfection and luciferase activity detection specifically comprises the following steps:

transfection: when the cell density in the 6-cm dish reached around 70%, cells were co-transfected with the mFSHR expression plasmid and pGL3-CRE-luciferase reporter plasmid, respectively. The amount of expression vector used was 400ng per well, pGL3-CRE-luciferase reporter plasmid 1600ng per well, transfection reagent jetPRIME 2. mu.L per well, a total of 200. mu.L of the mixture was made up with jetPRIME Buffer and left at room temperature for 10 min. Adding the transfection mixed solution into a 6-cm-centimeter culture dish, culturing in a CO2 incubator for 6h, replacing with a new DMEM low-sugar medium containing 5% FBS, and continuing to culture for 24 h.

Cells were plated in 96-well plates: cells were digested with tryptic digest and resuspended in DMEM-L medium containing 0.5% FBS at 180. mu.L per well (approx. 2X 10)⁴Individual cells) were added to a 96-well plate and cultured for 24 h.

Ligand treatment: the medium in the 96-well plate was aspirated, and 100. mu.L of serum-free DMEM low-sugar medium containing recombinant human FSH proteins at different concentrations was added to each well and cultured for 6 hours.

Cell lysis: 5 × Cell Culture Lysis Buffer was diluted 1 × with MilliQ-H2O, medium was aspirated, and 50 μ L of Cell lysate was added to each well quickly. After the cells are fully lysed, 15 mu L of lysate is taken from each hole and put into an enzyme label plate for measurement, 40 mu L of Luciferase substrate is added and mixed evenly, and the fluorescence value is measured in a German Berton multifunctional enzyme label instrument LB 941.

Effects of FSH hormones on intracellular cAMP signalling pathway referring to FIG. 2, after recombinant human FSH-treated cells in CHO-K1 cells expressing mouse FSHR, the mouse FSHR receptor was significantly activated at a concentration of 1ng/ml and had a stronger activation potential at a concentration of 10ng/ml, revealing that the system was working properly and could be used for assays for determining the activity of FSH proteins.

Example 4

In this example, we treated the cells expressing mouse FSHR with Conditioned Medium (CM) containing 2 μ l, 6 μ l and 20 μ l of panda FSH recombinant protein, respectively, and the results of the experiment are shown in fig. 3, which revealed that the culture medium with different conditioned medium contents can activate mouse FSHR receptor and that the activation effect is dose-dependent on the conditioned medium content, thus revealing that the panda FSH recombinant protein prepared in this example has biological activity and can specifically activate FSHR receptor.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> China panda protection research center

<120> recombinant expression vector of giant panda follicle-stimulating hormone, expression system and preparation method

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Ser Phe Ser Gly Phe Gly Asp Leu Glu Lys Ile Glu Ile Ser Gln Asn

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Leu His Glu Ile Arg Ile Glu Lys Ala Asn Asn Leu Leu Tyr Ile Asn

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Pro Glu Ala Phe Gln Asn Leu Pro Ser Leu Arg Tyr Leu Leu Ile Ser

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Asn Thr Gly Ile Lys His Leu Pro Ala Phe His Lys Ile Gln Ser Leu

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Gln Lys Val Leu Leu Asp Ile Gln Asp Asn Ile Asn Ile His Ile Ile

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Ala Arg Asn Ser Phe Met Gly Leu Ser Phe Glu Ser Val Ile Leu Trp

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Leu Asn Lys Asn Gly Ile Gln Glu Ile His Asn Cys Ala Phe Asn Gly

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Thr Gln Leu Asp Glu Leu Asn Leu Ser Asp Asn Asn Asn Leu Glu Glu

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Leu Pro Asp Asp Val Phe Gln Gly Ala Ser Gly Pro Val Val Leu Asp

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Ile Ser Arg Thr Lys Val Tyr Ser Leu Pro Asn His Gly Leu Glu Asn

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Leu Lys Lys Leu Arg Ala Arg Ser Thr Tyr Arg Leu Lys Lys Leu Pro

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Ser Leu Asp Lys Phe Val Met Leu Ile Glu Ala Ser Leu Thr Tyr Pro

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Ser His Cys Cys Ala Phe Ala Asn Trp Arg Arg Gln Thr Ser Glu Leu

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His Pro Ile Cys Asn Lys Ser Ile Ser Arg Gln Asp Ile Asp Asp Met

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Thr Gln Pro Gly Asp Gln Arg Val Ser Leu Val Asp Asp Glu Pro Ser

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Tyr Gly Lys Gly Ser Asp Met Leu Tyr Ser Glu Phe Asp Tyr Asp Leu

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Cys Asn Glu Phe Val Asp Val Thr Cys Ser Pro Lys Pro Asp Ala Phe

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Asn Pro Cys Glu Asp Ile Met Gly Tyr Asn Ile Leu Arg Val Leu Ile

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Trp Phe Ile Ser Ile Leu Ala Ile Thr Gly Asn Thr Thr Val Leu Val

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Val Leu Thr Thr Ser Gln Tyr Lys Leu Thr Val Pro Arg Phe Leu Met

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Cys Asn Leu Ala Phe Ala Asp Leu Cys Ile Gly Ile Tyr Leu Leu Leu

405 410 415

Ile Ala Ser Val Asp Ile His Thr Lys Ser Gln Tyr His Asn Tyr Ala

420 425 430

Ile Asp Trp Gln Thr Gly Ala Gly Cys Asp Ala Ala Gly Phe Phe Thr

435 440 445

Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu Ala Ala Ile Thr Leu

450 455 460

Glu Arg Trp His Thr Ile Thr His Ala Met Gln Leu Glu Cys Lys Val

465 470 475 480

Gln Leu Cys His Ala Ala Ser Ile Met Val Leu Gly Trp Ala Phe Ala

485 490 495

Phe Ala Ala Ala Leu Phe Pro Ile Phe Gly Ile Ser Ser Tyr Met Lys

500 505 510

Val Ser Ile Cys Leu Pro Met Asp Ile Asp Ser Pro Leu Ser Gln Leu

515 520 525

Tyr Val Met Ala Leu Leu Val Leu Asn Ala Leu Ala Phe Val Val Ile

530 535 540

Cys Gly Cys Tyr Thr His Ile Tyr Leu Thr Val Arg Asn Pro Asn Ile

545 550 555 560

Val Ser Ser Ser Arg Asp Thr Lys Ile Ala Lys Arg Met Ala Thr Leu

565 570 575

Ile Phe Thr Asp Phe Leu Cys Met Ala Pro Ile Leu Phe Phe Ala Ile

580 585 590

Ser Ala Ser Leu Lys Val Pro Leu Ile Thr Val Ser Lys Ala Lys Ile

595 600 605

Leu Leu Val Leu Phe Tyr Pro Ile Asn Ser Cys Ala Asn Pro Phe Leu

610 615 620

Tyr Ala Ile Phe Thr Lys Asn Phe Arg Arg Asp Phe Phe Val Leu Met

625 630 635 640

Ser Lys Phe Gly Cys Tyr Glu Val Gln Ala Gln Ile Tyr Lys Thr Glu

645 650 655

Thr Ser Ser Ile Thr His Asn Phe His Ser Arg Lys Asn Pro Cys Ser

660 665 670

Ser Ala Pro Arg Val Thr Asn Ser Tyr Val Leu Val Pro Leu Asn His

675 680 685

Ser Val Gln Asn

690

Claims (10)

1. A vector for recombinant expression of giant panda follicle stimulating hormone is characterized by comprising a first gene for coding a giant panda FSH α subunit and a second gene for coding a giant panda FSH β subunit, wherein the sequence of the first gene is shown as SEQ ID No.1, and the sequence of the second gene is shown as SEQ ID No. 2.

2. The vector for recombinantly expressing panda follitropin according to claim 1, wherein the vector further comprises a linking element;

preferably, the connecting element is a T2A element, and the sequence of the T2A element is shown as SEQ ID NO. 3.

3. A method for constructing a vector for recombinantly expressing panda follitropin according to any one of claims 1 to 2, comprising the steps of: synthesizing the first gene and the second gene.

4. The method of constructing according to claim 3, further comprising ligating the synthesized first and second genes and the T2A element into a plasmid pCDH to construct a vector for recombinant expression of giant panda follitropin containing the inserted first gene, T2A element and second gene.

5. The method of claim 4, wherein the plasmid pCDH is first digested simultaneously with EcoR I and BamH I to linearize the vector, then digested simultaneously with EcoR I and BamH I to linearize the insert, and the linearized insert is ligated to the linearized pCDH using T4 DNA ligase.

6. A eukaryotic cell expression system comprising the vector for recombinantly expressing panda follitropin according to any one of claims 1 to 2 or the vector constructed by the construction method according to any one of claims 3 to 5.

7. The eukaryotic cell expression system of claim 6, wherein the eukaryotic cell expression system is constructed by transferring the vector into a eukaryotic cell;

preferably, the eukaryotic cell is constructed from CHO-K1 cells.

8. A method for producing recombinant panda follitropin in a eukaryotic cell expression system, comprising culturing the eukaryotic cell expression system of claim 6.

9. The method according to claim 8, wherein the constructed vector for recombinant expression of giant panda follitropin is transferred into CHO-K1 cells to construct a eukaryotic cell expression system containing the vector for recombinant expression of giant panda follitropin.

10. The method according to claim 8, further comprising isolating and purifying the cultured eukaryotic cell expression system;

preferably, the method of isolation and purification comprises subjecting the cultured eukaryotic cell expression system to at least one of salting out, ion exchange chromatography separation and affinity chromatography separation.

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